A recent study that examined multiple strains of Campylobacter jejuni reported that HS:19, a serostrain that has been associated with the onset of Guillain-Barre syndrome, had unidentified labile, capsular polysaccharide (CPS) structures. In this study, we expand on this observation by using current glyco-analytical technologies to characterize these unknown groups. Capillary electrophoresis electrospray ionization MS and NMR analysis with a cryogenically cooled probe (cold probe) of CPS purified using a gentle enzymatic method revealed a hyaluronic acid-type [-4)-β-D-GlcA6NGro-(1-3)-β-D-GlcNAc-(1-]n repeating unit, where NGro is 2-aminoglycerol. A labile α-sorbofuranose branch located at C2 of GlcA was determined to have the L configuration using a novel pyranose oxidase assay and is the first report of this sugar in a bacterial glycan. A labile O-methyl phosphoramidate group, CH3OP(O)(NH2)(OR) (MeOPN), was found at C4 of GlcNAc. Structural heterogeneity of the CPS was due to nonstoichiometric glycosylation with sorbose at C2 of GlcA and the nonstoichiometric, variably methylated phosphoramidate group. Examination of whole bacterial cells using high-resolution magic angle spinning NMR revealed that the MeOPN group is a prominent feature on the cell surface for this serostrain. These results are reminiscent of those in the 11168 and HS:1 strains and suggest that decoration of CPS with nonstoichiometric elements such as keto sugars and the phosphoramidate is a common mechanism used by this bacterium to produce a structurally complex surface glycan from a limited number of genes. The findings of this work with the HS:19 serostrain now present a means to explore the role of CPS as a virulence factor in C. jejuni.

capsular polysaccharide, Campylobacter jejuni, phosphoramidate, high-resolution magic angle spinning (HR-MAS) NMR, sorbose

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,2	%aLSorf	61.5	104.3	79.2	76.1	79.2	62.9
3,6	x?GroN	61.5	53.9	61.5
3	bDGlcpA	101.0	73.8	73.8	79.0	75.1	170.7
2	Ac	175.0	23.5
4	%Subst	54.8
	bDGlcpN	100.7	56.1	75.8	74.2	75.5	61.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,2	%aLSorf	3.64-3.73	-	4.17	4.41	4.39	3.69-3.70
3,6	x?GroN	3.65-3.75	4.07	3.65-3.75
3	bDGlcpA	4.65	3.71	3.71	3.90	4.94	-
2	Ac	-	2.11
4	%Subst	3.77
	bDGlcpN	4.62	3.97	4.24	4.26	3.61	3.74-3.93


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,2	%aLSorf	61.5/3.64-3.73		79.2/4.17	76.1/4.41	79.2/4.39	62.9/3.69-3.70
3,6	x?GroN	61.5/3.65-3.75	53.9/4.07	61.5/3.65-3.75
3	bDGlcpA	101.0/4.65	73.8/3.71	73.8/3.71	79.0/3.90	75.1/4.94	
2	Ac		23.5/2.11
4	%Subst	54.8/3.77
	bDGlcpN	100.7/4.62	56.1/3.97	75.8/4.24	74.2/4.26	75.5/3.61	61.3/3.74-3.93

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@@H]1[C@@H](C(=O)NC(CO)CO)O[C@@H](O[C@@H]2[C@@H](NC(C)=O)[C@H](*)O[C@H](CO)[C@H]2OP(N)(=O)OC)[C@H](O[C@]2(CO)O[C@@H](CO)[C@@H](O)[C@@H]2O)[C@H]1O

707.576 g/mol (C₂₄H₄₂R₂N₃O₁₉P, R = next and previous repeats, not counted in mol weight)